illumina MiSeq System Manual
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illumina MiSeq System Manual

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MiSeq
System Guide
Document # 15027617 v04 Material # 20024228
July 2018
For Research Use Only. Not for use in diagnostic procedures.
ILLUMINA PROPRIETARY

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  • Page 1 MiSeq System Guide Document # 15027617 v04 Material # 20024228 ILLUMINA PROPRIETARY July 2018 For Research Use Only. Not for use in diagnostic procedures.
  • Page 2 MiSeq System Guide This document and its contents are proprietary to Illumina, Inc. and its affiliates ("Illumina"), and are intended solely for the contractual use of its customer in connection with the use of the product(s) described herein and for no other purpose.
  • Page 3 Added note to Reagent Compartment regarding required chiller temperature range. Added back MiSeq Reporter and removed references to Local Run Manager. Changed the name of the guide from the MiSeq Sequencing System Guide to the MiSeq System Guide. Minor text edits. Material # 20024228...
  • Page 4 Document # Date Description of Change Material # 20000262 September Changed the name of the guide from the MiSeq System User Document # 15027617 v01 2015 Guide to the MiSeq System Guide. New in MCS v2.6, added BaseSpace Onsite information.
  • Page 5 For information about these topics, see the MiSeq Reporter User Guide , part # 15028784, or the MiSeq Sample Sheet Quick Reference Guide , part # 15028392. Removed instructions for preparing custom primers. For more information, see Using Custom Primers on the MiSeq , part # 15041638.
  • Page 6 Removed instructions for denaturing and diluting DNA 2013 libraries and preparing an Illumina PhiX control. See Preparing DNA Libraries for Sequencing on the MiSeq , part # 15039740. Updated instrument wash instructions to add 25 ml 10% Tween 20 to 475 ml laboratory-grade water, instead of 500 ml laboratory-grade water.
  • Page 7 • Removed Sample Sheet Parameters section and sample sheet setup step in the workflow; Illumina recommends creating the sample sheet prior to sample preparation (See the MiSeq Sample Sheet Quick Reference Guide , part # 15028392 and the Illumina Experiment Manager User Guide , part # 15031335).
  • Page 8 • Analysis duration—Added when analysis exceeds two hours • Analysis Input Requirements—Listed manifest files as required for TruSeq Custom Amplicon libraries • Corrected HT1 tube size in MiSeq Reagent Kit Contents • Changed iCom references to MyIllumina Part # 15027617 Rev. A September...

  • Page 9: Table Of Contents

    Components MiSeq Concepts System Software Secondary Analysis Options Sequencing Analysis Viewer Required Disk Space MiSeq Reagent Kit Overview Chapter 2 Getting Started Start the MiSeq Customize System Settings Configure Notifications of BaseSpace Updates Set Email Preferences Set Default Folder Locations...
  • Page 10 MiSeq System Guide Appendix A Troubleshooting Introduction Bundle Logs for Troubleshooting Perform a System Check Pause or Stop a Run Raise Reagent Cartridge Sippers Manually Resolve Run Setup Errors Resolve RFID Read Failure Perform a Volume Test Measure Expected Wash Volumes...

  • Page 11: Chapter 1 Overview

    (SBS) technology with a revolutionary workflow that lets you go from DNA to analyzed data in as few as eight hours. The MiSeq integrates cluster generation, sequencing, and data analysis on a single instrument.

  • Page 12: Components

    Guide (document # 15049148) Components The MiSeq comprises a touch screen monitor, a status bar, a power button with adjacent USB ports, and three compartments. Document # 15027617 v04 Material # 20024228 For Research Use Only. Not for use in diagnostic procedures.
  • Page 13 F Reagent compartment—Contains reagents at proper temperatures, wash solutions, and a bottle for used reagents. A magnetic latch secures the reagent compartment door. The MiSeq interface guides you through the run setup steps using the touch screen monitor. Loading run components requires access to the reagent compartment and the flow cell compartment.

  • Page 14: Miseq Concepts

    The sample sheet can be provided during the run setup steps on the MiSeq. After the run begins, the sample sheet is renamed to SampleSheet.csv and copied to the run folders: MiSeqTemp, MiSeqOutput, and MiSeqAnalysis.

  • Page 15: System Software

    For more information on analysis workflows and manifest file formats, see the Local Run Manager Software Guide (document # 1000000002702) . For more information on sample sheets, see the MiSeq Sample Sheet Quick Reference Guide (document # 15028392) . System Software The instrument software suite includes integrated applications that perform sequencing runs, on-instrument analysis, and related functions.

  • Page 16: Secondary Analysis Options

    Status of BaseSpace connection (not connected shown) Secondary Analysis Options MiSeq sequencing data can be analyzed on the instrument computer using Local Run Manager, on a ™ networked server using BaseSpace Onsite, or on the cloud using BaseSpace. These applications produce information about alignment, variants, and contig assemblies for each genome requested and for each ™...
  • Page 17 BaseSpace and BaseSpace Onsite automatically disconnect from the MiSeq at the end of the run or as soon as all RTA analysis files have finished transfer. If the internet connection is interrupted, analysis files continue to upload after the connection is restored from the point when the interruption occurred.

  • Page 18: Sequencing Analysis Viewer

    SAV is universal to Illumina sequencing systems, most of which use an 8-lane flow cell. Some views include drop-down lists showing lanes 1–8. Because the MiSeq flow cell has a single lane, select All or Lane 1. For more information, see the Sequencing Analysis Viewer User Guide (document # 15020619) .
  • Page 19 Yellow Reagent Cartridge Overview The MiSeq reagent cartridge is a single-use consumable consisting of foil-sealed reservoirs prefilled with clustering and sequencing reagents sufficient for sequencing one flow cell. Each reservoir on the cartridge is numbered. Sample libraries are loaded onto the cartridge in position 17, which is labeled Load Samples.
  • Page 20 Reserved for custom Read 2 primer [Optional] NOTE For more information about using custom primers on the MiSeq reagent cartridge, see the MiSeq Custom Primers Guide (document # 15041638) . Document # 15027617 v04 Material # 20024228 For Research Use Only. Not for use in diagnostic procedures.

  • Page 21: Chapter 2 Getting Started

    Wait for the system to load, and then log on to the operating system. If necessary, consult your facility administrator for the user name and password. When the operating system is loaded, the MiSeq Control Software (MCS) launches and initializes the system automatically.

  • Page 22: Configure Notifications Of Basespace Updates

    Select Automatically check for new software updates on BaseSpace. Set Email Preferences MiSeq can be configured to send an email notification when RTA analysis is complete, when on-instrument secondary analysis is complete, or if a critical MiSeq software error occurs.

  • Page 23: User-Supplied Consumables

    MiSeq System Guide Output Folder— Sets the default location for analysis output files. Change the default output folder to a network location for sharing, and long-term storage. For more information, see Run Folders on page Recipe Folder—Sets the default location for recipes. Recipes are XML files that the software uses to perform the sequencing run.
  • Page 24 MiSeq System Guide Milli-Q water Super-Q water Molecular biology grade water Document # 15027617 v04 Material # 20024228 For Research Use Only. Not for use in diagnostic procedures.

  • Page 25: Chapter 3 Sequencing

    Run duration is based on the number of cycles performed. You can perform a paired-end run up to 2 x 301 sequencing cycles plus any Index Reads with MCS v2.3 or later. Additionally, run duration is based on the version of MiSeq reagents you are using and any performance enhancing upgrades installed on your instrument.
  • Page 26 MiSeq System Guide For example, a paired-end 300-cycle run performs two reads of 301 cycles (2 x 301) for a total of 602 cycles. At the end of the run, 2 x 300 cycles are analyzed. Document # 15027617 v04 Material # 20024228...

  • Page 27: Miseq Workflow

    Prepare the prefilled reagent cartridge for use. Denature and dilute libraries (does not apply to all library types). See Preparing Libraries for Sequencing on the MiSeq (document # 15039740) . Load the library mix onto the reagent cartridge in the designated reservoir.

  • Page 28: Thaw Reagent Cartridge

    Figure 10 Maximum Water Line Allow the reagent cartridge to thaw in the room temperature water bath until it is thawed completely. MiSeq v3 cartridges— ~ 60-90 minutes. MiSeq v2 cartridges— ~ 60 minutes. Remove the cartridge from the water bath and gently tap it on the bench to dislodge water from the base of the cartridge.

  • Page 29: Inspect The Reagent Cartridge

    Gently tap the cartridge on the bench to reduce air bubbles in the reagents. NOTE The MiSeq sipper tubes go to the bottom of each reservoir to aspirate the reagents, so it is important that the reservoirs are free of air bubbles.

  • Page 30: Set Up A Run Using Mcs

    MiSeq System Guide Figure 11 Load Libraries Proceed directly to the run setup steps using the MiSeq Control Software (MCS) interface. Set Up a Run Using MCS From the Home screen, select Manage Instrument. Select Reboot to reboot the system software.

  • Page 31: Clean The Flow Cell

    BaseSpace or BaseSpace Onsite. From the BaseSpace Options screen, select Use BaseSpace™. Selecting this option also stores the raw data from this run on the MiSeq. Select one of the following: Send all run data to BaseSpace Sequence Hub for remote monitoring and data analysis.
  • Page 32 MiSeq System Guide Using plastic forceps, grip the flow cell by the base of the plastic cartridge and remove it from the flow cell container. Figure 12 Remove Flow Cell Lightly rinse the flow cell with laboratory-grade water until both the glass and plastic cartridge are thoroughly rinsed of excess salts.

  • Page 33: Load The Flow Cell

    MiSeq System Guide Figure 15 Dry Flow Cell Dry excess alcohol with a lint-free lens cleaning tissue. Make sure that the flow cell ports are free of obstructions and that the gasket is well-seated around the flow cell ports. If the gasket appears to be dislodged, gently press it back into place until it sits securely around the flow cell ports.

  • Page 34: Load Reagents

    MiSeq System Guide Gently press down on the flow cell clamp to close it over the flow cell. As the flow cell clamp closes, alignment pins position the flow cell. An audible click indicates that the flow cell clamp is secure.
  • Page 35 NOTE Do not leave the reagent chiller door open for extended periods of time. Hold the reagent cartridge on the end with the Illumina label, and slide the reagent cartridge into the reagent chiller until the cartridge stops. Always use the reagent cartridge associated with the type of flow cell that you loaded. If the reagent cartridge is not compatible, a message appears on the screen.

  • Page 36: Start The Run

    MiSeq System Guide If the reagent cartridge is not compatible with the flow cell, a message appears. Select Back to load a compatible cartridge, or select Exit to return to the Home screen. Close the reagent compartment door. Select Next.

  • Page 37: Monitor The Run

    Pause a Run on page WARNING Make sure to close all files on the MiSeq before starting a run, and do not open files during a run. Monitor the Run During a run, monitor run detail using the Sequencing screen on the instrument. The Sequencing screen is view-only.
  • Page 38 1 in 10 Cluster Density (K/mm²)—Shows the number of clusters per square millimeter for the run. Clusters Passing Filter (%)—Shows the percentage of clusters passing filter based on the Illumina chastity filter, which measures quality. This data appears only after cycle 25.

  • Page 39: Perform A Post-Run Wash

    MiSeq System Guide For MiSeq run specifications, visit the MiSeq System specifications page on the Illumina website (www.illumina.com/systems/miseq/performance_specifications.ilmn). RTA Analysis Results The RTA analysis output from a sequencing run is a set of quality-scored base call files (*.bcl), which are generated from the raw image files.
  • Page 40 MiSeq System Guide Add 6 ml wash solution to each reservoir of the wash tray. b Add 350 ml wash solution to the 500 ml wash bottle. When the run is complete, select Start Wash. The software automatically raises the sippers in the reagent chiller.
  • Page 41 MiSeq System Guide dilution. b Add 50 µl of the 1:25 NaOCl dilution to 950 µl of laboratory-grade water in a MiSeq tube (part # MS- 102-9999). NOTE Using the correct concentration of NaOCl is important. Make sure to check the percentage of NaOCl on the product label.
  • Page 42 For additional environmental, health, and safety information, see the SDS at support.illumina.com/sds.html. 12 Slowly lower the sipper handle, making sure that the sippers lower into the wash bottle and waste bottle.

  • Page 43: Chapter 4 Maintenance

    Chapter 4 Maintenance Maintenance Frequency Maintenance Frequency for the VeriSeq PGS Workflow Perform a Maintenance Wash Perform a Standby Wash Manage Files Software Updates Shut Down the Instrument Maintenance Frequency Perform the following maintenance procedures at the recommended intervals. NOTE If performing the VeriSeq PGS workflow, make sure to follow maintenance frequency guidelines for VeriSeq PGS.

  • Page 44: Perform A Maintenance Wash

    MiSeq System Guide Table 5 Maintenance During Idle Mode (≥ 7 days unused) Activity Frequency Standby Wash Monthly Instrument Shutdown As needed Perform a Maintenance Wash Perform a maintenance wash every 30 days to ensure optimal performance. The maintenance wash takes approximately 90 minutes to complete. The wash includes a series of three wash steps that thoroughly flush the system.
  • Page 45 MiSeq System Guide Remove the waste bottle and discard the contents appropriately. Return the waste bottle to the reagent compartment. b Slowly lower the sipper handle, making sure that the sippers lower into the wash bottle and waste bottle. Close the reagent compartment door.

  • Page 46: Perform A Standby Wash

    MiSeq System Guide Slide the wash tray into the reagent chiller until it stops. Close the reagent chiller door. b Load the wash bottle and slowly lower the sipper handle, making sure that the sippers lower into the wash bottle and waste bottle.
  • Page 47 MiSeq System Guide Add 6 ml wash solution to each reservoir of the wash tray. b Add 350 ml wash solution to the 500 ml wash bottle. Load the wash tray and wash bottle onto the instrument: Open the reagent compartment door and reagent chiller door, and slide the used reagent cartridge or wash tray from the chiller.

  • Page 48: Manage Files

    Select OK. Upload Files The Upload command is available for Sample Sheets, Manifests, Genomes, and Recipes. If the MiSeq is not connected to a network, use this feature to upload files to the instrument computer from a USB drive. From the tab on the Manage Files screen, select Browse to navigate to files accessible to the instrument.

  • Page 49: Software Updates

    Update Software Automatically When software updates are available, the Update Available button appears on the Home screen. Otherwise, this button is not visible. Make sure your MiSeq is connected to a network with internet access to enable this option. From the Home screen, select Update Available.
  • Page 50 MiSeq System Guide Close the reagent compartment door. From the Home screen, select Manage Instrument. Select Shut Down. This command shuts down the software. Toggle the power switch to the OFF position. NOTE Any time that you turn off the instrument, wait a minimum of 60 seconds before turning the power switch back to the ON position.

  • Page 51: Appendix A Troubleshooting

    On the Manage Files screen, select the Bundle Logs tab. Select Browse to navigate to the location of the MiSeqOutput folder. Click in the blue box next to the run, and in the blue circle next to the bundle type requested by Illumina Technical Support.

  • Page 52: Perform A System Check

    Select Done. Pause or Stop a Run The MiSeq is designed to complete a run from beginning to end without user intervention. However, it is possible to pause a run or stop a run from the Sequencing screen. Document # 15027617 v04Material # 20024228...
  • Page 53 MiSeq System Guide Pause a Run You can temporarily pause a run before it has completed. For example, a run can be paused if you suspect that the waste bottle is full. Paused runs can be resumed. When you select Pause, the current command is completed before pausing the run and placing the flow cell in a safe state.

  • Page 54: Raise Reagent Cartridge Sippers Manually

    Log in to your MyIllumina account. Your name replaces the Sign In button on the toolbar. Hover over your name and select Account. In the My Tools column, click MiSeq Self-Service. On the MiSeq Self-Service page, enter the MiSeq serial number.

  • Page 55: Perform A Volume Test

    MiSeq System Guide Consumable Barcode Number Location Flow Cell Above the barcode on the flow cell container label. Flow cell barcode numbers begin with an A (standard), G (micro), or D (nano). Example: A0E61 PR2 Bottle Below the barcode on the PR2 bottle label.

  • Page 56: Measure Expected Wash Volumes

    The required temperature range of the reagent chiller is 2°C to 11°C. A sensor indicator shows the Sensor Indicators on page temperature of the reagent chiller. See If you receive an error message that the chiller is not in the specified temperature range, contact Illumina Technical Support. For more information on the reagent chiller, see...

  • Page 57: Appendix B Output Files And Folders

    Each run on the MiSeq generates 3 run folders, each with a specific purpose: D:\Illumina\MiSeqTemp—When the run begins, a temporary run folder is written to the local drive of the instrument computer and used as a working area for MCS and RTA. There is no need to access the MiSeqTemp folder.
  • Page 58 MiSeq System Guide RunInfo.xml—Contains high-level run information, such as the number of reads and cycles in the sequencing run, and whether a read is indexed. Folders The folders that are copied to the output and analysis folders include the following folders generated during the sequencing run: <Run folder name>\Config—Contains configuration files for the run.
  • Page 59 During the sequencing run, the single lane of the flow cell is imaged in small imaging areas called tiles. All MiSeq flow cells have a single lane, but the number of tiles differ depending on the type of flow cell you are using.
  • Page 60 Standard Flow Cell MiSeq Reagent Kits, v2 1-14 1101 through 1114 1-14 Bottom 2101 through 2114 Micro Flow Cell MiSeq Reagent Micro Kits, v2 1101 through 1104 Bottom 2101 through 2104 Nano Flow Cell MiSeq Reagent Nano Kits, v2 Top only 1101...

  • Page 61: Index

    6 customer support 55 sensors 6 cycles in a read 15 icons, blinking 5 idling the instrument 36 Illumina Proactive monitoring service 12 InterOp files 5 deleting files and folders 38 InterOp folder 48 disk space IP address 46...
  • Page 62 MiSeq System Guide Local Run Manager 1 troubleshooting 44 overview 7 RTAcomplete.txt 49 run duration 15 run folder definition 4 run folders maintenance wash 34 contents 47 manifest file managing 38 copying to instrument 38 naming 47 definition 4 primary analysis files 49...
  • Page 63 MiSeq System Guide RFID 44 run-specific files for 41 Universal Copy Service 5 updating software 39 VeriSeq PGS workflow flow cell 9 maintenance frequency 33 replicate analysis locally 11 secondary analysis 6 volume test 45 warnings 5 wash volumes 46...
  • Page 64 MiSeq System Guide Document # 15027617 v04Material # 20024228 For Research Use Only. Not for use in diagnostic procedures.

  • Page 65: Technical Assistance

    Other countries +44.1799.534000 Safety data sheets (SDSs)—Available on the Illumina website at support.illumina.com/sds.html. Product documentation—Available for download in PDF from the Illumina website. Go to support.illumina.com, select a product, then select Documentation & Literature. Document # 15027617 v04Material # 20024228...
  • Page 66 MiSeq System Guide Document # 15027617 v04Material # 20024228 For Research Use Only. Not for use in diagnostic procedures.

  • Page 67: Document # 15027617 V04 Material # 20024228

    Document # 15027617 v04 Material # 20024228 Illumina 5200 Illumina Way San Diego, California 92122 U.S.A. +1.800.809.ILMN (4566) +1.858.202.4566 (outside North America) techsupport@illumina.com www.illumina.com For Research Use Only. Not for use in diagnostic procedures. © 2018 Illumina, Inc. All rights reserved.

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